Human rhinoviruses (HRVs) are the most frequent cause of acute upper respiratory tract infections in humans and are usually associated with the common cold. Common colds caused by HRV occur throughout the year, with peaks of incidence in the autumn and spring, are one of the main reasons for absences from work and school, which have major economic impact. Rhinoviruses can also cause lower respiratory tract infections resulting in severe disease in children, in the elderly and in immunosuppressed patients.
The HRVs, which include over 100 different serotypes, are small, non-enveloped, positive (+)-strand RNA viruses, HRVs are one of the six genera of Picornaviridae, which also includes enteroviruses (EVs). Reverse transcription-polymerase chain reaction (RT-PCR) has been developed in the past few years for the detection of the HRVs in clinical specimens (see, e.g., Billaud et al. (2003) J. Virol. Methods 108: 223-228; Blomqvist et al. (1999) J. Clin, Microbiol. 37:2813-2816; Kares et al. (2003) J Clin Virol. 2004 February; 29(2):99-104; Loens et al. (2003) J. Clin. Microbiol. 41: 1971-1976; Savolainen et al. (2002) J. General Virol. 83: 333-340; Steininger et al. (2001) J. Clin. Microbiol. 39: 129-133). Most of these RT-PCR methods take advantage of the conserved sequences in the 5′ noncoding region of the picornavirus genome.
The ability to detect HRV specifically—and particularly avoiding false positives that can result due to the relatedness of HRV and EV—is important to both diagnosis and selection of appropriate available therapy. Specific assays for HRV are also important for development of new drugs. For example, it is critical for clinical trial design that the participants be correctly identified as having an HRV infection where the trial is designed to evaluate a drug for use in treatment of HRV infections. Moreover, in other clinical trials, it may be important to exclude individuals infected with HRV. Further, the HRV detection assays must be simple to perform, provide easily interpreted results, and be relatively inexpensive to make them practical for use.
Conventional methods of differentiation of HRVs from EVs has been done either by virus neutralization assay, by selection with HRV-specific primer pairs, by distinguishing the amplification products of the two viruses based on differences in size, by sequencing the amplification products and comparing the sequence to known HRV and EV sequences, or by hybridization using HRV or EV-specific probes. These approaches can be time-consuming, expensive, and/or require a skilled technician who has experience in interpreting assay results accurately.
There remains a need in the field for methods for detecting RVs in a manner that is rapid, sensitive and specific, particularly with respect to the ability to distinguish an RV from an EV.
The present invention addresses these needs.
Literature
Literature of interest includes:
Billaud et al. (2003) “Detection of rhinovirus and enterovirus in upper respiratory tract samples using a multiplex nested PCR” J. Virol. Methods 108: 223-228; Blomqvist et al. (1999) “Rapid detection of human rhinoviruses in nasopharyngeal aspirates by a microwell reverse transcription-PCR-hybridization assay.” J. Clin. Microbiol. 37: 2813-2816; Hyypia T., et al. (1998). Molecular diagnosis of human rhinovirus infections: comparison with virus isolation. J. Clin Microbiol. 36: 2081-2083; Kares et al. (2003) “Real-time PCR for rapid diagnosis of entero- and rhinovirus infections using LightCycler” J Clin Virol. 2004 February; 29(2):99-104; Loens et al. (2003) “Improved detection of rhinoviruses by nucleic acid sequence-based amplification after nucleotide sequence determination of the 5′ noncoding regions of additional rhinovirus strains.” J. Clin. Microbiol. 41: 1971-1976; Santti J., et al. (1997). Comparison of PCR primer pairs in the detection of human rhinoviruses in nasopharyngeal aspirates. J. Virol. Methods 66: 139-147; Savolainen et al. (2002) “Genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70.” J. General Virol. 83: 333-340; Steininger et al. (2001) “Early detection of acute rhinovirus infections by rapid reverse transcription-PCR assay.” J. Clin. Microbiol. 39: 129-133; and U.S. Publication No. US2002/0142294.